| Alzheimer’s disease(AD) is an irreversible brain disorder characterized by progressive cognitive decline and neurodegeneration of brain regions that induce learning and memory declining as well as daily living ability decreasing and abnormal behavior. The pathological characteristic includes senile plaques and neurofibrillary tangles. Aβ has been recognized the core factor in AD pathopoiesis. More studies about effect of Aβ on inducing AD focus mainly on neuron degeneration, inflammation and oxidative stress. Recent studies have found nerve dysfunction and loss of neuron correlate well with pathological accumulation of soluble Aβ. Microglia is the representative of immunoreactive cell in CNS, playing an important role in inflammation. Some study has shown that when MG is activated by accumulated Aβ, the expresstion of receptors on MG are upregulated, the phagocytic and clearance function of MG to Aβ strengthen gradually. However, as Aβ increase to a particular high concentration, the scavenging to Aβ may become down. But it is still unclear what is the exact relation between Aβ concentration and the change of MG phagocytic clearance. We speculate it might be related with the expression of phagocytosis receptor SRA on MG. Recent studies have proved that antibody blocking TLR2 can prevent NF-κB expressing and TNF-α、IL-1β releasing, indicating TLR2 may initiate NF-κB signal routing and promote inflammation, but some other researches give a opposite result that loss of TLR2 has no relationship with reduced pro-inflammatory cytokines. RAGE, a receptor on MG, is upregulated in AD human brain, but it’s role in AD mechanism has not been known lately. BV2, also called glioblastoma, is extensively used as microglia in researching AD field. Different concentration Aβ were co-incubated with BV2 for 48 h to observe morphologic change of BV2,the expression of SRA、TLR2、RAGE as well as TNF-α、IL-1β. This experiment aims at futher analyzing the link between different concentration Aβ and BV2 morphology, founction, effect of SRA、TLR2、RAGE in AD to state the role of MG and offer dependable clinical targeted therapies for AD treatment.Objective: To explore the effect of different concentration Aβ to BV2 livability and the change of BV2 clearance to Aβ. To detect the expression of SRA, TLR2, RAGE, NF-κB protein and TNF-α、IL-1β releasing and anlyze the connection between different concentration Aβ and the change of BV2 morphology and function. To state the role of SRA, TLR2, RAGE and to offer dependable clinical targeted therapies for AD treatment.Method: Normal cultured cells were co-incubated with different concentration Aβ(0, 1, 5,10, 15μmol/L)for 48 h. The morphology change of BV2 was observed under microscope; MTT was used to detect the BV2 livability; ELISA was used to test IL-1β, TNF-α and residual Aβ in cell supernatant. ICC and WB were used to measure the expression of SRA, TLR2, RAGE, NF-κB protein.Results: 1 BV2 morphology: After 48 h co-incubation, BV2 in 0μmol/L group were quiescent, and soma was small, showing longitypical or triangular, small and dense nucleus, vimineously branched burr. BV2 in Aβ group showed cnidocil rebound, and pseudopodium extends, amoeboid. With the growing concentration of Aβ25-35, the change of BV2 morphology became obviously phagocyte-like. 2 BV2 viability: MTT result showed that BV2 livability in 1,5μmol/L groups are repectively(97.89%±1.98)%,(82.89%±1.73)%, both higher than 80%; However, BV2 livability in 10,15μmol/L groups were(47.26%±2.91)%,(43.75%±0.21) %, a significant difference compared with 5μmol/L group (P<0.05). 3 IL-1β、TNF-α expression and residual Aβ in cell supernatant: ELISA result showed that the level of IL-1β in 10,15μmol/L groups were respectively(54.8333±2.9145) pg/m L,(59.2778±1.4510)pg/m L, obviously higher than 5μmol/L group(13.5000±1.3298)pg/m L; level of TNF-α in 10,15μmol/L groups were respectively(72.0739±6.1020) pg/m L,(84.7128±7.2849)pg/m L, markly higher than 5μmol/L group(66.1067±4.6830)pg/m L. The clearance to Aβ in 5μmol/L group is(0.8693±0.0096)μmol/L, higher than that in 1μmol/L group(0.7190±0.0045)μmol/L, while the clearance to Aβ in 10,15μmol/L groups were respectively(0.4781±0.0059) μmol/L,(0.2238±0.0021) μmol/L, distinctly lower than that in 5μmol/L group.All of the comparation have statistical significance(P<0.05). 4 Immunocytochemistry: ICC result showed that SRA positive area in 10,15μmol/L groups were respectively(0.41±0.17),( 0.30±0.30),extremely lower than that in 5μmol/L group(0.84±0.22)(P<0.05);Howeve, for the positive area of TLR2( 0.39±0.02, 0.42±0.01), RAGE( 0.41±0.10, 0.50±0.04), and intranuclear NF-κB(0.30±0.01, 0.39±0.04)protein in 10,15μmol/L groups were all obviously higher than their parallel protein in 5μmol/L group(TLR2:0.18±0.06, RAGE: 0.29±0.08,intranuclear NF-κB: 0.19±0.07), all groups had statistical significance(P<0.05). 5 Western Blotting: WB result showed that expression of SRA protein in 10,15μmol/L groups were respectively(1.13±0.49),(1.08±0.30), extremely lower than that in 5μmol/L group(2.14±0.12)(P<0.05); Howeve, for the expression of TLR2( 0.88±0.14, 0.91±0.31),RAGE( 0.54±0.17, 0.59±0.21), and intranuclear NF-κB(1.63±0.01, 1.84±0.36)protein in 10,15μmol/L groups were all obviously higher than their parallel protein in 5μmol/L group(TLR2: 0.45±0.15, RAGE: 0.26±0.18,intranuclear NF-κB: 1.04±0.05), all groups had statistical significance(P<0.05).Conclusion: The clearance ability of BV2 to Aβ enhanced gradually with a gradual increase in the concentration of Aβ(1,5μmol/L).However, the clearance ability of BV2 reduced when Aβ concentration comes to 10μmol/L; This mechanism might have relationship with SRA upregulation,TLR downregulation,RAGE downregulation and activated NF-κB signal routing, which mediate the releasing of IL-1β, TNF-α. |
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